Figure 4.

The results of anti-NMDAR autoAb tests were confirmed by double labeling. The three experimental protocols utilizing the IIFT kit (as illustrated in Figures 3A–C) were compared. The degree of green/red colocalization, represented by R_coloc, was indicated beneath each dual-color merged image. Top—Patient A: The degrees of colocalization between a diluted plasma sample from patient A and the mouse anti-NR1 mAb improved after incorporating glutaraldehyde fixation following sample labeling (R_coloc ~0 → ~0.17). Middle—False-positive: A diluted blood sample showed positive signals by the standard single-labeling protocol (left images), and were later deemed “false-positive” by both double-labeling tests (right panels). This sample with “false-positive” results failed to colocalize with heterologously-expressed NMDAR by either tests illustrated in Figures 3B,C. Bottom—anti-NMDAR-negative: No green fluorescence was shown in the Tester BIOCHIPs by single or double labeling with an anti-NMDAR-negative plasma sample. Yellow arrowheads pointed to sites of colocalization of green and red fluorescence (overlay in yellow-orange color). Scale bars, 20 μm.